Multiplex immunochromatography test strip with unified detection limit for detecting four aminoglycoside drugs and application of multiplex immunochromatography test strip

Abstract

The invention discloses a multi-immunochromatography test strip with unified detection limit for detecting four aminoglycoside drugs and application of the multi-immunochromatography test strip. The test strip comprises a water absorption plate, a detection pad, a colloidal gold pad, a sample pad and a PVC (polyvinyl chloride) bottom plate, wherein a gold-labeled antibody is fixed on the surface of the colloidal gold pad, and when a sample flows through the colloidal gold pad, the gold-labeled antibody is dissolved and serves as a tracer molecule to migrate along with a liquid phase; four detection areas T1, T2, T3 and T4 and a quality control line area C are arranged on the detection pad, a kanamycin antigen, a gentamicin antigen, a neomycin antigen and a spectinomycin antigen are sprayed on the four detection areas respectively, and a goat anti-mouse antibody is sprayed on the quality control line area C. According to the present invention, the expensive reagent and the base material are not required, the production cost is low, the operation is simple, the professional training is not required, the detection efficiency is high, the detection cost is low, the multiple immunochromatography test strip is used for simultaneously detecting kanamycin, gentamicin, neomycin and spectinomycin, and the detection limits can achieve 5 ng / mL.

Description

Technical Field

[0001] This invention relates to a multiplex immunochromatographic test strip with uniform detection limits for four aminoglycoside drugs and its application, belonging to the field of rapid pesticide residue detection. Background Technology

[0002] Aminoglycosides (AGs) are broad-spectrum antibiotics widely used in animal therapy and clinical medicine. Their misuse or non-compliant use can lead to drug residues in animal-derived foods. Long-term low-dose intake can lead to drug resistance risks, threatening public health. Furthermore, aminoglycosides have ototoxicity and nephrotoxicity, posing a higher risk to sensitive populations such as children and pregnant women.

[0003] Traditional microbiological assays determine antibiotic residues based on their inhibitory effects on microorganisms, but the results are easily affected by many factors, leading to poor sensitivity and specificity. Chromatography, as the most commonly used instrumental analytical method for detecting pesticide and veterinary drug residues in food, boasts high sensitivity and accuracy, but its high cost makes it unsuitable for rapid on-site testing. With the diversified evolution of science and technology and the deepening interaction and exchange between different disciplines, affinity-based analytical methods are continuously being innovated.

[0004] Colloidal gold immunochromatography is a simple, rapid, and sensitive immunological detection technique. Based on the principle of antigen-antibody specific binding chromatography, the target antigen or antibody is immobilized on a chromatographic membrane, with colloidal gold nanoparticles pre-loaded as signal probes in the binding pad. After the sample solution is added, a colorimetric signal is rapidly generated in the reaction zone for qualitative and quantitative detection of the target analyte.

[0005] Currently, colloidal gold immunochromatographic technology for detecting aminoglycosides suffers from poor detection sensitivity and inconsistent detection limits when detecting multiple aminoglycosides. This leads to unreliable results for most colloidal gold immunochromatographic test strips used to detect multiple aminoglycosides, resulting in false negatives, false positives, or misinterpretations. Therefore, there is an urgent need to provide a multiplex immunochromatographic test strip with high detection sensitivity and a uniform detection limit to meet the need for simultaneous screening of multiple common drugs, thereby improving regulatory efficiency. Summary of the Invention

[0006] To address the shortcomings of current methods for detecting aminoglycoside drugs, this paper presents a multiplex immunochromatographic test strip with a unified detection limit for four aminoglycoside drugs, along with its preparation method and application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution; A multiplex immunochromatographic test strip with uniform detection limits for four aminoglycoside drugs is disclosed. The test strip comprises five parts: an absorbent plate, a test pad, a colloidal gold pad, a sample pad, and a PVC base plate. The sample pad is the sample application area. A gold-labeled antibody is immobilized on the surface of the colloidal gold pad. When the sample flows through it, the gold-labeled antibody dissolves and migrates with the liquid phase as a tracer molecule. The test pad has five independent areas: four detection areas (T1, T2, T3, and T4) and a control area (C). Kanamycin antigen, gentamicin antigen, neomycin antigen, and spectinomycin antigen are sprayed onto the four detection areas (T1, T2, T3, and T4), respectively. Goat anti-mouse antibody is sprayed onto the control area (C). The test strip consists of four layers from bottom to top. The first layer is the bottom layer, which is composed of a PVC base plate. The second layer is the detection pad, which is placed on top of the bottom layer. The third layer is composed of a colloidal gold pad and an absorbent pad. The top layer is the sample pad. The PVC base plate serves as the bottom layer, fully supporting the second, third, and top layers. The second layer is the detection pad; the third layer consists of a colloidal gold pad and an absorbent pad, which are not in contact with each other and are located at both ends of the detection pad, partially overlapping it. The gap between the colloidal gold pad and the absorbent pad is divided into four detection areas (T1, T2, T3, and T4) and a quality control line area C; the sample pad of the third layer is located above the colloidal gold pad and partially overlaps with it. Preferably, the sample pad and colloidal gold pad are made of glass fiber; the absorbent plate is made of cellulose; and the detection pad is a nitrocellulose membrane.

[0008] The sample pad requires pretreatment before use. The pretreatment method is as follows: wash the sample pad with pure water, dry it at 37°C, soak it in the treatment solution for 1 hour, and then dry it at 37°C to obtain the pretreated sample pad. The components of the treatment solution are: 0.01 M PBS buffer solution containing a final concentration of 0.2% (w / v) Tween-20, 1% (w / v) sucrose, and 1% (w / v) BSA. Preferably, the colloidal gold pad surface is immobilized with gold-labeled antibodies, which are composed of gold-labeled antibodies against kanamycin, gentamicin, neomycin, and spectinomycin in a volume ratio of 1:1 to 1.5:1 to 1.5:1; the concentration of the kanamycin antigen, gentamicin antigen, neomycin antigen, and spectinomycin antigen sprayed is 0.2-1.2 mg / mL; the concentration of goat anti-mouse antibody sprayed in control area C is 0.5 mg / mL.

[0009] More preferably, the spray concentration of the kanamycin antigen is 0.6 mg / mL, the spray concentration of the gentamicin antigen is 0.8 mg / mL, the spray concentration of the neomycin antigen is 0.8 mg / mL, and the spray concentration of the spectinomycin antigen is 0.4 mg / mL. The preparation steps for the gold-labeled antibody are as follows:

[0010] Step 1: Screening of hybridoma cell lines (1) Screening of positive mice: First, kanamycin, gentamicin, spectinomycin and neomycin immunogens were mixed with an equal volume of Freund's complete adjuvant to obtain four immune mixtures, which were then injected into mice for the first immunization. Preferably, in step 1 (1), the concentrations of the immunogens kanamycin, gentamicin, spectinomycin, and neomycin are all 2 mg / mL; the injection dose is 4~6 μg / g based on the mouse's body weight; and the first immunization cycle is 21 days. After the initial immunization, kanamycin, gentamicin, spectinomycin and neomycin immunogens were mixed with an equal volume of Freund's incomplete adjuvant to obtain four booster immunization mixtures, which were then injected into mice for booster immunization. Preferably, the injection dose of the booster immunization mixture in step 1 (1) is 2~3 μg / g based on the mouse's body weight; the booster immunization is performed three times: the first and second booster immunizations are performed over a period of 21 days, and the third booster immunization is performed over a period of 7 days; After booster immunization, blood was collected from the tail vein of mice, and the titer and inhibition rate of mouse serum were detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Mice with the highest titer and inhibition rate were selected for shock immunization. Kanamycin immunogen, gentamicin immunogen, spectinomycin immunogen, and neomycin immunogen were mixed with an equal volume of physiological saline to obtain four shock immunization mixtures, which were injected into the peritoneal cavity of mice for shock immunization. Preferably, the injection dose of the shock immunization in step 1 (1) is based on the mouse body weight: 1~1.2 μg / g; the shock immunization cycle is 3 days.

[0011] (2) Cell fusion: (2-1) After shock immunization, mouse spleens were ground into powder and mixed with myeloma cells SP2 / 0. The mixture was centrifuged and the supernatant was discarded. The precipitate was collected. Then, preheated polyethylene glycol (PEG) solution was added to the precipitate. After mixing and standing, RPMI-1640 medium was added in batches. After standing, the mixture was centrifuged again, the supernatant was discarded, the precipitate was collected, and HAT medium was added to the precipitate. The mixture was then mixed to obtain mixed cells. Preferably, in step 1 (2-1), the ratio of spleen powder to myeloma cells SP2 / 0 is 5-10g:1ml; the centrifugation conditions are: centrifugation at 1000 r / min for 5 min; the concentration of polyethylene glycol (PEG) solution is 1g / mL; the preheating temperature is 37℃; and the mixing and standing time is 1-2 min. The ratio of the amount of precipitate, polyethylene glycol (PEG) solution, RPMI-1640 medium, and HAT medium used is 0.5~1g:1 mL:20ml:10~20ml; the standing temperature is 37℃ and the time is 5 min; the conditions for centrifugation again are: centrifugation at 800 r / min for 5 min. (2-2) The mixed cells were transferred to a 96-well cell culture plate for culture. During the culture, half of the HAT medium was replaced at certain intervals. After the culture was completed, the HAT medium was removed and replaced with an equal volume of HT medium. The mixture was mixed evenly to obtain the culture medium. The supernatant was obtained after centrifugation. Preferably, the culture in step 1 (2-2) is carried out in a carbon dioxide incubator at a temperature of 37 ℃; the culture period is 6 days, with a certain interval of 3 days.

[0012] (3) Cell screening: First, add PBS solution to the 96-well microplate, then add the supernatant obtained in step (2-2), and use the ic-ELISA method to screen out positive wells; then take the supernatant of the positive wells and dilute it with antibody diluent to obtain diluted cell supernatant; Finally, the standard solution corresponding to the immunogen was mixed with the diluted cell supernatant and detected by ic-ELISA. Four cell lines were finally obtained: the cell line obtained by immunization with kanamycin immunogen was designated as hybridoma cell line 4E9, the cell line obtained by immunization with gentamicin immunogen was designated as hybridoma cell line 4A4, the cell line obtained by immunization with spectinomycin immunogen was designated as hybridoma cell line 4B2, and the cell line obtained by immunization with neomycin immunogen was designated as hybridoma cell line 5A8. Preferably, in step 1 (3), the concentration of the PBS solution is 0.01 M; the volume ratio of the PBS solution, supernatant, and standard solution is 1:1:1; the antibody diluent is: 0.85 g gelatin and 0.15 g bovine serum albumin dissolved in 1 L of PBS solution; the concentration of the standard solution is 1 mg / ml; the standard solution includes kanamycin standard solution, gentamicin standard solution, spectinomycin standard solution, and neomycin standard solution; the absorbance of the diluted cell supernatant is in the range of 1.5-2.0.

[0013] Step 2: Preparation of monoclonal antibodies (1) Obtaining ascites: First, sterile paraffin oil was injected into the peritoneal cavity of mice to reduce immune rejection; then, the hybridoma cells obtained in step 1 were injected into the peritoneal cavity of mice. After a certain period of time, the mice's abdomens swelled and their mental state was poor. Ascites fluid was extracted, and the supernatant was obtained by centrifugation, which is the processed ascites fluid for later use. Preferred: In step 2 (1), the amount of sterile paraffin oil injected is 1 ml, and the injection cycle is 7 days; the hybridoma cells are hybridoma cell line 4E9, hybridoma cell line 4A4, hybridoma cell line 4B2, or hybridoma cell line 5A8; the cell density of the injected hybridoma cells is 1×10⁻⁶. 6 Cell density; injection time is 6-10 days, centrifugation conditions are: centrifugation at 5000 r / min for 10 min; (2) Ascites purification: Add acetate buffer to the treated ascites obtained in step (1), and add HCl solution to adjust the pH value to obtain an ascites sample; then add octanoic acid dropwise to the ascites sample, and after standing, centrifuge to obtain the supernatant. Preferred: In step 2 (2), the volume ratio of ascites fluid to acetate buffer is 1:2, and the pH value is adjusted to 4.3-4.7; the dependence of octanoic acid on ascites fluid sample is 30-35 μL: 1 mL; the standing condition is 4℃ for 2 h; the centrifugation condition is 4℃, centrifuged at 8000~10000 rpm for 10~20 min.

[0014] (3) Add PBS solution to the supernatant obtained in step (2), adjust the pH with NaOH solution to obtain a mixture, add saturated ammonium sulfate solution dropwise to the mixture, let it stand, centrifuge to collect the precipitate, redissolve it with PBS solution, and finally dialyze to obtain a monoclonal antibody solution; Depending on the type of hybridoma cells injected, monoclonal antibody solutions for kanamycin, gentamicin, neomycin, and spectinomycin were obtained.

[0015] Preferably, in step 2 (3), the volume ratio of the supernatant to the PBS solution is 10:1:1, the concentration of the PBS solution is 0.1M; the pH value is adjusted to 7; the volume ratio of the mixture to the saturated ammonium sulfate solution is 1:1, and the standing conditions are 4℃ for 2h; the centrifugation conditions are: 4℃, centrifugation at 8000~10000 rpm for 10~20min; the dialysis conditions are: dialysis at 4℃ for 72h; and the concentration of the monoclonal antibody solution is 1mg / mL.

[0016] Step 3: Preparation of gold-labeled antibodies (1) Preparation of colloidal gold solution: After boiling ultrapure water, keep it at a gentle boil, then add chloroauric acid solution under stirring conditions, stir for the first time, add sodium citrate solution after stirring, and continue stirring after the solution color changes from yellow to red. After stirring, turn off the heating and magnetic stirring, and cool to room temperature to obtain colloidal gold solution (store at 4℃ for later use). Preferably, in step 3 (1), the volume ratio of ultrapure water, chloroauric acid solution, and sodium citrate solution is 10000:3:2; wherein the mass concentration of chloroauric acid solution is 10% and the mass concentration of sodium citrate solution is 20%; the first stirring time is 5-8 min, and the stirring time is 15-20 min.

[0017] (2) Conjugation of colloidal gold particles with monoclonal antibodies: Take the colloidal gold solution prepared in step 3 (1), add K2CO3 to adjust the pH value of the solution, then add the monoclonal antibody solution prepared in step 2, mix by shaking, then add BSA blocking solution for blocking, after blocking, centrifuge to collect the precipitate, add resuspension solution for resuspending, and obtain gold-labeled antibody. Monoclonal antibodies include KAN, GEN, NEO, and SPE monoclonal antibodies. Depending on the type of monoclonal antibody (including KAN, GEN, NEO, and SPE), gold-labeled antibodies of KAN, GEN, NEO, or SPE are obtained respectively.

[0018] Preferably, in step 3 (2), the ratio of colloidal gold solution, monoclonal antibody solution, BSA blocking solution and resuspension is 2 mL: 15-20 μg: 200 μL: 200 μL, the mass concentration of BSA blocking solution is 10%, the concentration of K2CO3 is 0.1 M, and the pH of the solution is adjusted to 8.0-8.5; the blocking time is 1 h; the centrifugation conditions are: 4℃, 12000 r / min for 15 min; the resuspension is: PBS solution containing 1% (w / v) sucrose, 2% (w / v) BSA and 0.02% (v / v) sodium azide.

[0019] More preferably, the concentration of kanamycin monoclonal antibody in the gold-labeled kanamycin antibody is 10 μg / mL, the concentration of gentamicin monoclonal antibody in the gold-labeled gentamicin antibody is 12 μg / mL, the concentration of neomycin monoclonal antibody in the gold-labeled neomycin antibody is 10 μg / mL, and the concentration of spectinomycin monoclonal antibody in the gold-labeled spectinomycin antibody is 12 μg / mL.

[0020] The pH of the solution is adjusted by the amount of K2CO3 added. Specifically, the amounts added to each 1 mL of colloidal gold solution are: 10 μg of kanamycin monoclonal antibody and 12 μL of K2CO3; 12 μg of gentamicin monoclonal antibody and 8 μL of K2CO3; 10 μg of neomycin monoclonal antibody and 10 μL of K2CO3; and 12 μg of spectinomycin monoclonal antibody and 8 μL of K2CO3.

[0021] The multiplex immunochromatographic test strip of the present invention can achieve a detection limit of 5 ng / mL when used for the simultaneous detection of kanamycin, gentamicin, neomycin and spectinomycin. Beneficial effects:

[0022] This invention provides a method for preparing a multiplex immunochromatographic test strip capable of simultaneously detecting four aminoglycoside drugs, including kanamycin, gentamicin, spectinomycin, and neomycin. The invention first obtains four hybridoma cell lines through immunogenic screening using the immunogens of kanamycin, gentamicin, spectinomycin, and neomycin. Then, monoclonal antibodies against the aminoglycoside drugs (kanamycin, gentamicin, spectinomycin, and neomycin) are purified from ascites fluid and prepared as gold-labeled antibodies. This results in a multiplex immunochromatographic test strip with a uniform detection line. The invention effectively improves the detection performance of the test strip by optimizing the pH during coupling, the amount of antibody labeling, and the concentration of the coating antigen on the T line. The test strip provided by this invention is simple to operate, highly sensitive, and has a uniform detection limit of 5 ng / mL for all four aminoglycoside drugs. The multiplex detection results are consistent with the single-detection results, meeting the needs of rapid on-site screening. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the assembly of a colloidal gold immunochromatographic test strip. Figure 1 A) Schematic diagram of the principle of colloidal gold immunochromatographic test strip ( Figure 1 B) and schematic diagram of reading results from colloidal gold immunochromatographic test strip ( Figure 1 C).

[0024] Figure 2 This is a schematic diagram of the multilayer structure of a colloidal gold immunochromatographic test strip.

[0025] Figure 3 The image shows the screening results of four hybridoma cell lines. Figure 3 A represents the screening results of hybridoma cell line 4E9. Figure 3 B represents the screening results for hybridoma cell line 4A4. Figure 3 C represents the screening results of hybridoma cell line 4B2 and Figure 3 D represents the screening result of hybridoma cell line 5A8.

[0026] Figure 4 Here is a characterization diagram of colloidal gold particles, in which Figure 4 A is a real photograph of the colloidal gold particle solution. Figure 4 B is a transmission electron microscope (TEM) image of colloidal gold particles. Figure 4 C is the ultraviolet absorption spectrum of colloidal gold particles.

[0027] Figure 5The image shows the results of multiplex immunochromatographic assay strips detecting the four aminoglycosides in milk. Figure 5 A indicates that milk contains KAN. Figure 5 B indicates that milk contains GEN. Figure 5 C indicates that milk contains NEO. Figure 5 D indicates that milk contains SPE. Figure 5 E indicates that the milk contains four aminoglycosides (KAN, GEN, NEO, and SPE); lines 1-5 in the figure represent the C line, the KAN detection line, the GEN detection line, the NEO detection line, and the SPE detection line, respectively. Figure 5 Each AD test strip contains 5 strips, with spiked concentrations of 0, 0.5, 1, 2.5 and 5 ng / mL from left to right. Figure 5 E contains two test strips, with spiked concentrations of 0 and 5 ng / mL from left to right. Detailed Implementation

[0028] The technical solutions of the present invention will be further described below through specific embodiments. It should be understood that these embodiments are preferred solutions of the present invention and are intended to aid in understanding the technical content of the present invention, rather than limiting the scope of protection of the present invention. Without departing from the basic concept of the present invention, various substitutions, equivalent improvements, or combinations can be made by those skilled in the art, and all such substitutions, equivalent improvements, or combinations should be considered to fall within the scope of protection of the present invention.

[0029] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0030] The immunogens described in this invention include immunogens of kanamycin (KAN), gentamicin (GEN), spectinomycin (SPE), and neomycin (NEO), all purchased from Sangon Biotech (Shanghai) Co., Ltd.; kanamycin standard solution, gentamicin standard solution, spectinomycin standard solution, and neomycin standard solution were all purchased from Sinopharm Chemical Reagent Co., Ltd.; goat anti-mouse antibody was purchased from Thermo Fisher Scientific, USA, and the goat anti-mouse secondary antibody used in this embodiment is a goat anti-mouse secondary antibody. Example 1:

[0031] Step 1: Screening of hybridoma cell lines (1) Screening of positive mice: SPF-grade, 6-8 week old female BALB / c mice (provided by the Animal Experiment Center of Jiangsu University) were selected for the immunization experiment, with four mice in each group. First, the immunogen (immunogen of kanamycin, gentamicin, spectinomycin or neomycin) was diluted with physiological saline to 2 mg / mL. Then, 150 uL of the diluted immunogen and an equal volume of Freund's complete adjuvant (FCA) were injected into two syringes respectively. The two syringes were connected with a connecting tube and pushed back and forth until obvious resistance was felt. At this time, the immunogen and Freund's adjuvant were fully mixed and emulsified to obtain four immunization mixtures, which were injected into the mice for the first immunization. At the time of the first immunization, each mouse was subcutaneously injected with 100 μg of immunogen, and the first immunization cycle was 21 days. In the subsequent booster immunization phase, the injection dose was reduced to half of the initial immunization dose (i.e., 50 μg of immunogen), and the injection method remained subcutaneous. At the same time, Freund's complete adjuvant was replaced with Freund's incomplete adjuvant (FIA), resulting in four booster immunization mixtures, which were injected into mice for booster immunization. Three booster immunizations were administered: the first and second booster immunizations were administered over a 21-day period, and the third booster immunization was administered over a 7-day period. Seven days later, blood was collected from the tail vein of mice, and the serum titer and inhibition rate were determined using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Based on the test results, mice with high titers and good inhibition were selected for a pulse immunization. This pulse immunization involved mixing the immunogen with physiological saline and administering it via intraperitoneal injection at a dose of 25 μg per mouse. Cell fusion experiments were performed three days later.

[0032] (2) Cell fusion: Mouse spleen was ground into powder, and the spleen powder was mixed with myeloma cells SP2 / 0 (purchased from Gibco, USA) at a ratio of 8:1. The mixture was centrifuged at 1000 r / min for 5 min, the supernatant was discarded, and the precipitate was gently shaken to prepare for fusion. During fusion, 1 mL of preheated polyethylene glycol (PEG) solution (concentration 1 g / mL) was slowly dripped along the wall of the centrifuge tube in the first minute while shaking the tube to mix the cells; the mixture was allowed to stand for 1 min; 2 mL, 4 mL, and 8 mL of RPMI-1640 medium were added sequentially in the third, fourth, and fifth minutes to mix the cells, and the fusion was completed. The RPMI 1640 medium in the centrifuge tube was added to 20 mL, sealed with a sealing film, and allowed to stand at 37℃ for 5 min. Then, the mixture was centrifuged at 800 r / min for 5 min, and the supernatant was discarded. Subsequently, HAT medium was slowly added along the wall of the centrifuge tube to mix with the precipitate (i.e., the fused cells) to obtain mixed cells. The mixed cells were transferred to 96-well cell culture plates, 200 μL per well, and incubated in a CO2 incubator at 37 °C. On the third day of incubation, half of the culture medium in each well was aspirated and discarded, and an equal volume of preheated HAT medium at 37 °C was added to the remaining medium. On the sixth day of incubation, the culture medium in each well was aspirated and discarded, and replaced with an equal volume of HT medium at 37 °C. The mixture was thoroughly mixed to obtain the culture medium, which was then centrifuged to obtain the supernatant. (3) Cell screening: Add 0.01 M PBS solution to each well of the packaged 96-well ELISA plate, 50 μL. Then add the supernatant obtained in step (2) to each well of the 96-well ELISA plate containing PBS solution, 50 μL, and screen for positive wells using ic-ELISA. Take the supernatant corresponding to the positive wells and dilute it with antibody diluent (composition: 0.85 g gelatin and 0.15 g bovine serum albumin dissolved in 1 L PBS solution) to make its absorbance value in the range of 1.5-2.0. Add 50 μL of standard solution (antigen) to a new packaged 96-well ELISA plate, and then add the diluted cell supernatant. Detect the inhibition effect using ic-ELISA. The screening results are as follows. Figure 3 As shown, four cell lines were finally obtained through screening, denoted as hybridoma cell line 4E9 ( Figure 3 A, obtained by immunization with the kanamycin immunogen), hybridoma cell line 4A4 ( Figure 3 B, obtained by immunization with gentamicin immunogen), hybridoma cell line 4B2 ( Figure 3 C, obtained by immunogen immunization with spectinomycin) and hybridoma cell line 5A8 ( Figure 3 D, obtained through immunotherapy with neomycin immunogen.

[0033] Step 2: Preparation of monoclonal antibodies (1) Ascites fluid collection: Healthy BALB / c mice around 12 weeks old (provided by the Animal Experiment Center of Jiangsu University) were selected, and each mouse was injected intraperitoneally with 1 mL of sterile paraffin oil to reduce immune rejection and create a rapid growth environment for subsequent implantation of hybridoma cells. The injection cycle was 7 days; 7 days later, each mouse was injected with 1×10 6 To determine the optimal cell density, hybridoma cells were injected into the peritoneal cavity of mice. Mice were observed for 6 to 10 days post-injection. If abdominal swelling was significant and the mice exhibited poor mental status, ascites fluid was aspirated. The ascites fluid was centrifuged at 5000 r / min for 10 min, and the supernatant was the treated ascites fluid. The supernatant was collected, aliquoted into centrifuge tubes, and stored at -20℃.

[0034] (2) Ascites purification: Add acetate buffer to the ascites obtained in step (1) at a volume ratio of 1:2 (ascites:buffer), and add HCl solution dropwise to adjust the pH of the ascites sample to 4.3; add octanoic acid dropwise to the adjusted ascites sample at a ratio of 30 μL / mL. Let stand at 4℃ for 2 h, then centrifuge at 8000~10000 rpm for 10 min at 4℃, and take the supernatant after centrifugation. Add one-tenth of the volume of 0.1M PBS solution to the supernatant, adjust the pH to about 7 with NaOH solution, add saturated ammonium sulfate solution dropwise at a volume ratio of 1:1, and let stand at 4℃ for 2 h. Centrifuge at 10000 rpm for 20 min at 4℃, take the precipitate after centrifugation, redissolve it with 0.01M PBS solution, dialyze at 4℃ for 72 h, and finally obtain the monoclonal antibody solution with a concentration of 1 mg / ml by dialyzing. Depending on the type of hybridoma cells injected, this embodiment yields monoclonal antibody solutions for kanamycin, gentamicin, neomycin, and spectinomycin, respectively.

[0035] Step 3: Sensitivity and specificity testing of monoclonal antibodies: This invention uses the crossover rate (CR) to represent specificity and employs the ic-ELISA method to detect the IC of structural analogs of standards. 50 The crossover rate is calculated using the following formula:

[0036] Table 1. Results of KAN antibody cross-linking determination

[0037] Table 2. Results of GEN antibody cross-linking determination

[0038] Table 3. Results of NEO antibody cross-linking determination

[0039] Table 4. Results of SPE antibody and cross-linking assay

[0040] Note: In Tables 1-4, the first compound is the test standard, and the following ones are structural analogs of the test standard.

[0041] As shown in Tables 1-4, the monoclonal antibodies against kanamycin, gentamicin, neomycin, and spectinomycin obtained in this embodiment all possess specificity. Example 2:

[0042] (1) Preparation of colloidal gold particles: Add 1.3 L of ultrapure water to a 2 L round-bottom flask, place the flask in a heating mantle and heat until the water boils for 5 min, then pour out the water. Rinse with ultrapure water 5 times, and observe whether there are water droplets inside the flask. If there are water droplets, continue rinsing.

[0043] Add 1 L of ultrapure water to a clean round-bottom flask. Adjust the power to maintain a gentle boil, ensuring no obvious bubbles on the flask walls. Turn on the magnetic stirrer and adjust the speed appropriately. Then add 0.3 mL of 10% chloroauric acid solution (ensuring the amount of chloroauric acid added is 0.03% of 1 L of ultrapure water). Stir for 5 min, then add 2 mL of 20% sodium citrate solution. After the solution color changes from yellow to colorless to black to red, continue stirring for 15 min. Turn off the heating and magnetic stirrer, remove the flask, and cool to obtain a colloidal gold solution for later use. Characterize the colloidal gold solution as follows: Figure 4 As shown, the prepared colloidal gold solution is red ( Figure 4 A) Under transmission electron microscopy, the colloidal gold particles were observed to be approximately 17 nm in size, exhibiting relatively uniform size and good dispersibility. Figure 4 B). Figure 4 C is the ultraviolet absorption spectrum of colloidal gold. As can be seen from the figure, the prepared colloidal gold solution has an ultraviolet absorption peak at 522 nm.

[0044] (2) Coupling of colloidal gold particles with monoclonal antibodies: Take 2 mL of the colloidal gold solution prepared in step (1) and add 0.1 M K2CO3 to adjust the pH of the solution to 8.0-8.5. Then add 15-20 μg of monoclonal antibody solution and mix on a shaker for 45 min. Then add 200 μL of 10% BSA blocking solution to block for 1 h. After blocking, centrifuge at 12000 r / min at 4℃ for 15 min in a high-speed refrigerated centrifuge, discard the supernatant, add 200 μL of resuspension to the precipitate and resuspend. Depending on the type of monoclonal antibody (including KAN, GEN, NEO and SPE), obtain gold-labeled antibody solutions of KAN, GEN, NEO or SPE respectively, and store at 4℃ in the dark. Example 3:

[0045] S1. The sample pads need to be washed with pure water, dried at 37°C, and then soaked in the treatment solution (treatment solution: 0.01 M PBS buffer solution containing 0.2% (v / v) Tween-20, 1% (w / v) sucrose and 1% (v / v) BSA) for 1 hour and then dried at 37°C to obtain the pretreated sample pads.

[0046] The gold-labeled antibodies corresponding to kanamycin, gentamicin, spectinomycin, and neomycin were mixed at a volume ratio of 1:1:1:1 and sprayed onto a glass cellulose membrane to obtain a colloidal gold pad. like Figure 2 As shown, the test strip comprises five parts: an absorbent plate (cellulose material), a detection pad (nitrocellulose membrane), a colloidal gold pad (glass fiber), a sample pad (glass fiber), and a PVC base plate. The sample pad is the sample application area. A gold-labeled antibody is immobilized on the surface of the colloidal gold pad. When the sample flows through, the gold-labeled antibody dissolves and migrates with the liquid phase as a tracer molecule. The detection pad has five independent areas: four detection areas (T1, T2, T3, T4) and a control area C. Kanamycin antigen, gentamicin antigen, neomycin antigen, and spectinomycin antigen are sprayed onto the four detection areas (T1, T2, T3, T4), respectively. Goat anti-mouse antibody is sprayed onto the control area C. The spray concentration of kanamycin antigen is 0.6 mg / mL, the spray concentration of gentamicin antigen is 0.8 mg / mL, the spray concentration of neomycin antigen is 0.8 mg / mL, and the spray concentration of spectinomycin antigen is 0.4 mg / mL. The test strip consists of four layers from bottom to top. The first layer is the bottom layer, which is composed of a PVC base plate. The second layer is the detection pad, which is placed on top of the bottom layer. The third layer is composed of a colloidal gold pad and an absorbent pad. The top layer is the sample pad. The PVC base plate serves as the bottom layer, fully supporting the second, third, and top layers. The second layer is the detection pad; the third layer consists of a colloidal gold pad and an absorbent pad, which are not in contact with each other and are located at both ends of the detection pad, partially overlapping it. The gap between the colloidal gold pad and the absorbent pad is divided into four detection areas (T1, T2, T3, and T4) and a quality control line area C; the sample pad of the third layer is located above the colloidal gold pad and partially overlaps with it. S2. The antigens of kanamycin, gentamicin, spectinomycin, and neomycin are sprayed onto the detection lines (T lines) of the nitrocellulose membrane (NC membrane), and the goat anti-mouse antibody that can bind to the four monoclonal antibodies prepared is sprayed onto the control lines (C lines) of the nitrocellulose membrane (NC membrane) to obtain the test pad.

[0047] Specifically, the nitrocellulose membrane streaking procedure is as follows: The antigens of the four aminoglycoside drugs (KAN, GEN, NEO, and SPE) are sequentially sprayed onto the nitrocellulose membrane to form T-lines, including four T-line regions (T1-T4), each T-line region not overlapping. T1 is KAN, T2 is GEN, T3 is NEO, and T4 is SPE. Goat anti-mouse antibodies that can bind to the four monoclonal antibodies prepared in Example 2 are sprayed onto the nitrocellulose membrane to form C-lines, resulting in the detection pad. The concentration of the goat anti-mouse secondary antibody finally streaked onto the C-lines is 0.5 mg / mL.

[0048] S3. Assemble the sample pad and colloidal gold pad prepared in step S1, the detection pad prepared in step S2, and the conventional components of the chromatography test strip (absorbent plate and sample pad) in the order of absorbent plate, detection pad, colloidal gold pad and sample pad from top to bottom on the PVC base plate to obtain the multiplex immunochromatographic test strip.

[0049] The absorbent plate should overlap the test pad by 2 mm at both ends to ensure smooth liquid flow on the test strip. After drying, store at 4°C away from light.

[0050] A schematic diagram of the assembled chromatography strip is shown below. Figure 1 As shown in Figure A, during use, the sample to be tested should be added to the sample pad at one end of the band. Under capillary action, the sample moves forward. When it moves to the T-line region where the fixed antibody is located, the target analyte in the sample will specifically bind to the antibody in that region and be retained, forming a visible red band. The color intensity is positively correlated with the concentration of the target analyte.

[0051] Meanwhile, the goat anti-mouse secondary antibody in line C will bind to the gold-labeled antibody to form a red band. The color development of this band can verify the validity of the experiment; if line C develops color, it indicates that the test strip experiment is normal. Figure 1 B).

[0052] like Figure 1 As shown in C, the four lines T1-T4 represent the regions of four fixed antigens. If no red line is displayed in the corresponding region, it means that there is antigen in the target object in the region that is not displayed in red, and the test result is positive. If all regions corresponding to T1-T4 are displayed in red, it means that there are no corresponding four antigens in the target object, and the result is negative.

[0053] In subsequent experiments, the C line, T1 line, T2 line, T3 line and T4 line on the chromatography strip were numbered 1-5 from top to bottom. Example 4:

[0054] The colloidal gold immunochromatographic test strip of this invention employs a competitive assay, suitable for the detection of small molecule antigens (drugs, pesticides, veterinary drugs, peptides). When a test sample containing the target substance passes through the colloidal gold pad, the gold-labeled antibody specifically binds to the target substance in the sample. The more target substance present in the sample, the more gold-labeled antibody binds to it, and the less free gold-labeled antibody remains. Consequently, fewer gold-labeled antibodies can bind to the antigen immobilized on the detection line, resulting in a lighter T-line color.

[0055] (1) Optimization of pH value of gold-labeled antibody: pH was adjusted with K2CO3 and the amount of K2CO3 added was used to control pH. 0, 2, 4, 6, 8, 10 and 12 μL of 0.1 M K2CO3 solution were added to 1 mL of colloidal gold solution and mixed thoroughly. Then 10 μg of antibody was added to each solution and the mixture was stirred at an appropriate speed for 45 min on a shaker. The solution color change was observed and the group with no significant color change and the least amount of K2CO3 solution added was determined to be the optimal amount. The results are shown in Table 5. The optimal amounts of K2CO3 for the four gold-labeled antibodies KAN, GEN, NEO and SPE were 12 μL, 8 μL, 10 μL and 8 μL, respectively.

[0056] Table 5. Color changes of solutions under different pH conditions

[0057] (2) Optimization of antibody labeling amount for gold-labeled antibodies: According to the results in Table 5, the corresponding volume of 0.1 M K2CO3 solution was added to 1 mL of colloidal gold solution to achieve the optimal pH. Then, monoclonal antibody solution was added. The final concentrations of monoclonal antibodies were 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 12 μg / mL, 14 μg / mL and 16 μg / mL. The solution was mixed at a suitable speed for 45 min on a shaker. By observing the color change of the solution, the group with no significant color change and the least amount of antibody added was determined as the optimal amount. The optimal amounts of monoclonal antibodies for the four gold-labeled antibodies KAN, GEN, NEO and SPE were 10 μg / mL, 12 μg / mL, 10 μg / mL and 12 μg / mL, respectively.

[0058] Table 6. Color changes of solution under different antibody concentrations

[0059] (3) Optimization of the concentration of coating antigens (i.e., antigens fixed in the T-line region, including KAN, GEN, NEO, and SPE) on the T-line: 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg / mL of coating antigens were sprayed onto the T-line position of the nitrocellulose membrane and dried in a 37°C oven for 12 h. If the concentration of coating antigens on the T-line is too low, the color development will be indistinct and the result cannot be judged by the naked eye; if the concentration of coating antigens on the T-line is too high, the color development will be too deep, and the colloidal gold will easily aggregate. The color development reaction was carried out under the optimal pH and antibody labeling conditions, and the group with good and uniform color development was selected as the optimal concentration of coating antigens on the T-line. The preferred concentrations of coating antigens corresponding to the KAN, GEN, NEO, and SPE colloidal gold immunochromatographic test strips were 0.6, 0.8, 0.8, and 0.4 mg / mL, respectively.

[0060] (4) Establishment of the multiplex immunochromatographic test strip method: Add different concentrations (0, 0.5, 1, 2.5 and 5 ng / mL) of KAN, GEN, NEO and SPE standards to milk (any commercially available) as test solutions; add the test solution and the gold-labeled antibody corresponding to the standard in the test solution to the microwells, insert the sample pad of the colloidal gold immunochromatographic test strip prepared in Example 3 into the microwells, and perform a color reaction for 10 min.

[0061] The results are as follows Figure 5 As shown, the color change of the T line on the multiplex immunochromatographic strip was observed with the naked eye. All visible detection lines were 5 ng / mL. Figure 5 Each AD test strip contains 5 strips arranged in a row, with spiked concentrations of 0, 0.5, 1, 2.5 and 5 ng / mL from left to right. Figure 5 A contains the KAN standard. Figure 5 B contains GEN standard products. Figure 5 C contains NEO standard products. Figure 5 The added substance (D is SPE standard) does not interfere with each other, and the simultaneous presence of multiple T lines and multiple gold-labeled antibodies does not affect the detection sensitivity of the multiplex immunochromatographic test strip. When milk contains four aminoglycoside drugs (KAN, GEN, NEO, and SPE) at the same time (all at a concentration of 5 ng / mL), the color of the T line on the multiplex immunochromatographic test strip disappears. This indicates that the test strip prepared by this invention can accurately detect the four aminoglycoside drugs KAN, GEN, NEO, and SPE, and the detection limit can reach 5 ng / mL. The detection limit is uniform and meets the national limit standard.

[0062] The embodiments described above are merely some embodiments of the present invention. The accompanying drawings show preferred embodiments of the present invention, but these do not limit the scope of this application. Their purpose is to enable those skilled in the art to gain a more thorough and comprehensive understanding of the disclosure of this application. All equivalent embodiments based on the content of this application's specification and drawings, whether directly or indirectly applied to other related technical fields, are within the scope of patent protection of this application.

Claims

1. A multiplex immunochromatographic test strip with uniform detection limits for four aminoglycoside drugs, characterized in that, The steps are as follows: The test strip comprises five parts: an absorbent plate, a detection pad, a colloidal gold pad, a sample pad, and a PVC base plate. The sample pad is the sample application area. The surface of the colloidal gold pad is immobilized with gold-labeled antibodies. When the sample flows through it, the gold-labeled antibodies dissolve and migrate with the liquid phase as tracer molecules. The detection pad has five independent areas: four detection areas (T1, T2, T3, and T4) and a control area (C). Kanamycin antigen, gentamicin antigen, neomycin antigen, and spectinomycin antigen are sprayed onto the four detection areas (T1, T2, T3, and T4), respectively. Goat anti-mouse antibody is sprayed onto the control area (C). The test strip consists of four layers from bottom to top. The first layer is the bottom layer, which is composed of a PVC base plate. The second layer is the detection pad, which is placed on top of the bottom layer. The third layer is composed of a colloidal gold pad and an absorbent pad. The top layer is the sample pad. The PVC base plate serves as the bottom layer, fully supporting the second, third, and top layers. The second layer is the detection pad; the third layer consists of a colloidal gold pad and an absorbent pad, which are not in contact with each other and are located at both ends of the detection pad, partially overlapping it. The gap between the colloidal gold pad and the absorbent pad is divided into four detection areas (T1, T2, T3, and T4) and a quality control line area C; the sample pad of the third layer is located above the colloidal gold pad and partially overlaps with it. The preparation steps of the gold-labeled antibody are as follows: Step 1: Screening of hybridoma cell lines (1) Screening of positive mice: First, kanamycin, gentamicin, spectinomycin and neomycin immunogens were mixed with an equal volume of Freund's complete adjuvant to obtain four immune mixtures, which were then injected into mice for the first immunization. After the initial immunization, kanamycin, gentamicin, spectinomycin and neomycin immunogens were mixed with an equal volume of Freund's incomplete adjuvant to obtain four booster immunization mixtures, which were then injected into mice for booster immunization. After booster immunization, blood was collected from the tail vein of mice, and the titer and inhibition rate of mouse serum were detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Mice with the highest titer and inhibition rate were selected for shock immunization. Kanamycin immunogen, gentamicin immunogen, spectinomycin immunogen, and neomycin immunogen were mixed with an equal volume of physiological saline to obtain four shock immunization mixtures, which were injected into the peritoneal cavity of mice for shock immunization. (2) Cell fusion: (2-1) After the shock immunization, the spleen of the mouse was ground into powder and mixed with myeloma cells SP2 / 0. The mixture was centrifuged and the supernatant was discarded. The precipitate was collected. Then, preheated polyethylene glycol (PEG) solution was added to the precipitate. After mixing and standing, RPMI-1640 medium was added in batches. After standing, the mixture was centrifuged again, the supernatant was discarded, the precipitate was collected, and HAT medium was added to the precipitate. The mixture was then mixed to obtain the mixed cells. (2-2) The mixed cells were transferred to a 96-well cell culture plate for culture. During the culture, half of the HAT medium was replaced at certain intervals. After the culture was completed, the HAT medium was removed and replaced with an equal volume of HT medium. The mixture was mixed evenly to obtain the culture medium. The supernatant was obtained after centrifugation. (3) Cell screening: First, add PBS solution to the 96-well microplate, then add the supernatant obtained in step (2-2), and use the ic-ELISA method to screen out positive wells; then take the supernatant of the positive wells and dilute it with antibody diluent to obtain diluted cell supernatant; Finally, the standard solution corresponding to the immunogen was mixed with the diluted cell supernatant and detected by ic-ELISA. Four cell lines were finally obtained: the cell line obtained by immunization with kanamycin immunogen was designated as hybridoma cell line 4E9, the cell line obtained by immunization with gentamicin immunogen was designated as hybridoma cell line 4A4, the cell line obtained by immunization with spectinomycin immunogen was designated as hybridoma cell line 4B2, and the cell line obtained by immunization with neomycin immunogen was designated as hybridoma cell line 5A8. Step 2: Preparation of monoclonal antibodies (1) Obtaining ascites: First, sterile paraffin oil was injected into the peritoneal cavity of mice to reduce immune rejection; then, the hybridoma cells obtained in step 1 were injected into the peritoneal cavity of mice. After a certain period of time, the mice's abdomens swelled and their mental state was poor. Ascites fluid was extracted, and the supernatant was obtained by centrifugation, which is the processed ascites fluid for later use. (2) Ascites purification: Add acetate buffer to the treated ascites obtained in step (1), and add HCl solution to adjust the pH value to obtain an ascites sample; then add octanoic acid dropwise to the ascites sample, and after standing, centrifuge to obtain the supernatant. (3) Add PBS solution to the supernatant obtained in step (2), adjust the pH with NaOH solution to obtain a mixture, add saturated ammonium sulfate solution dropwise to the mixture, let it stand, centrifuge to collect the precipitate, redissolve it with PBS solution, and finally dialyze to obtain a monoclonal antibody solution; Depending on the type of hybridoma cells injected, monoclonal antibodies against kanamycin, gentamicin, neomycin, and spectinomycin were obtained. Step 3: Preparation of gold-labeled antibodies (1) Preparation of colloidal gold solution: After boiling ultrapure water, keep it at a gentle boil, then add chloroauric acid solution under stirring conditions and stir for the first time. After stirring, add sodium citrate solution. After the solution color changes from yellow to red, continue stirring. After stirring, turn off the heating and magnetic stirring, and cool to room temperature to obtain colloidal gold solution. (2) Conjugation of colloidal gold particles with monoclonal antibodies: Take the colloidal gold solution prepared in step 3 (1), add K2CO3 to adjust the pH value of the solution, and then add the monoclonal antibody solution prepared in step 2. After shaking and mixing, add BSA blocking solution for blocking. After blocking, centrifuge to collect the precipitate and add resuspension solution for resuspending to obtain gold-labeled antibody.

2. The multiplex immunochromatographic test strip for detecting four aminoglycoside drugs with uniform detection limits according to claim 1, characterized in that, The sample pad and colloidal gold pad are made of glass fiber; the absorbent plate is made of cellulose material; and the detection pad is a nitrocellulose membrane. The sample pad requires pretreatment before use. The pretreatment method is as follows: wash the sample pad with pure water, dry it at 37°C, soak it in the treatment solution for 1 hour, and then dry it at 37°C to obtain the pretreated sample pad. The components of the treatment solution are: 0.01 M PBS buffer solution containing a final concentration of 0.2% (w / v) Tween-20, 1% (w / v) sucrose, and 1% (w / v) BSA. The colloidal gold pad is immobilized with gold-labeled antibodies, which are composed of gold-labeled antibodies against kanamycin, gentamicin, neomycin, and spectinomycin in a volume ratio of 1:1 to 1.5:1 to 1.5:

1. The concentration of the kanamycin antigen, gentamicin antigen, neomycin antigen, and spectinomycin antigen sprayed is 0.2-1.2 mg / mL. The concentration of goat anti-mouse antibody sprayed in control area C is 0.5 mg / mL.

3. The multiplex immunochromatographic test strip for detecting four aminoglycoside drugs with uniform detection limits according to claim 2, wherein the spray concentration of kanamycin antigen is 0.6 mg / mL, the spray concentration of gentamicin antigen is 0.8 mg / mL, the spray concentration of neomycin antigen is 0.8 mg / mL, and the spray concentration of spectinomycin antigen is 0.4 mg / mL.

4. A multiplex immunochromatographic test strip for detecting four aminoglycoside drugs with uniform detection limits according to claim 1, characterized in that, In step 1 (1), the concentrations of the immunogens kanamycin, gentamicin, spectinomycin, and neomycin were all 2 mg / mL; the injection dose was 4-6 μg / g based on the mouse's body weight; the first immunization cycle was 21 days. The injection dose of the booster immunization mixture was 2-3 μg / g based on the mouse's body weight; the booster immunization was performed three times: the first and second booster immunizations were administered over a period of 21 days, and the third booster immunization was administered over a period of 7 days. The injection dose for shock immunization is 1~1.2 μg / g based on the mouse's body weight; the shock immunization cycle is 3 days.

5. A multiplex immunochromatographic test strip for detecting four aminoglycoside drugs with uniform detection limits according to claim 1, characterized in that, In step 1 (2-1), the ratio of spleen powder to myeloma cells SP2 / 0 is 5-10g: 1ml; the centrifugation conditions are: centrifugation at 1000 r / min for 5 min; the concentration of polyethylene glycol solution is 1g / mL; the preheating temperature is 37℃; and the mixing and standing time is 1-2 min. The ratio of the amount of precipitate, polyethylene glycol solution, RPMI-1640 medium, and HAT medium used is 0.5~1g:1 mL:20ml:10~20ml; the standing temperature is 37℃ and the time is 5 min; the conditions for centrifugation again are: centrifugation at 800 r / min for 5 min.

6. A multiplex immunochromatographic test strip for detecting four aminoglycoside drugs with uniform detection limits according to claim 1, characterized in that, In step 1 (2-2), the culture is carried out in a carbon dioxide incubator at a temperature of 37°C; the culture cycle is 6 days, with a certain interval of 3 days. In step 1 (3), the concentration of the PBS solution is 0.01 M; the volume ratio of the PBS solution, supernatant, and standard solution is 1:1:1; the antibody diluent is: 0.85 g gelatin and 0.15 g bovine serum albumin dissolved in 1 L of PBS solution; the concentration of the standard solution is 1 mg / ml; the standard solution includes kanamycin standard solution, gentamicin standard solution, spectinomycin standard solution, and neomycin standard solution; the absorbance of the diluted cell supernatant is in the range of 1.5-2.

0.

7. A multiplex immunochromatographic test strip for detecting four aminoglycoside drugs with uniform detection limits according to claim 1, characterized in that, In step 2(1), the amount of sterile paraffin oil injected is 1 ml, and the injection cycle is 7 days; the hybridoma cells are hybridoma cell line 4E9, hybridoma cell line 4A4, hybridoma cell line 4B2, or hybridoma cell line 5A8; the cell density of the injected hybridoma cells is 1×10⁻⁶. 6 Cell density; injection time is 6-10 days, centrifugation conditions are: centrifugation at 5000 r / min for 10 min; In step 2 (2), the volume ratio of ascites fluid to acetate buffer is 1:2, and the pH value is adjusted to 4.3-4.7; the dependence of octanoic acid on ascites fluid sample is 30-35 μL: 1 mL; the standing conditions are 4℃ for 2 h; the centrifugation conditions are 4℃, centrifuged at 8000~10000 rpm for 10~20 min. In step 2 (3), the volume ratio of supernatant to PBS solution is 10:1, the concentration of PBS solution is 0.1M; adjust the pH value to 7; the volume ratio of mixture to saturated ammonium sulfate solution is 1:1, and the standing condition is 4℃ for 2h; the centrifugation condition is 4℃, centrifuged at 8000~10000 rpm for 10~20min; the dialysis condition is 4℃ for 72h; the concentration of monoclonal antibody solution is 1mg / mL.

8. The multiplex immunochromatographic test strip for detecting four aminoglycoside drugs with uniform detection limits according to claim 1, characterized in that, In step 3 (1), the volume ratio of ultrapure water, chloroauric acid solution, and sodium citrate solution is 10000:3:2; the mass concentration of chloroauric acid solution is 10%, and the mass concentration of sodium citrate solution is 20%; the first stirring time is 5-8 min, and the stirring time is 15-20 min. In step 3(2), the volume ratio of colloidal gold solution, monoclonal antibody solution, BSA blocking solution and resuspension solution is 2 mL: 15-20 μg: 200 μL: 200 μL, the mass concentration of BSA blocking solution is 10%, the concentration of K2CO3 is 0.1 M, and the pH of the solution is adjusted to 8.0-8.5; the blocking time is 1 h; the centrifugation conditions are: 4℃, 12000 r / min for 15 min. The resuspension was a PBS solution containing 1% (w / v) sucrose, 2% (w / v) BSA and 0.02% (v / v) sodium azide.

9. The multiplex immunochromatographic test strip according to claim 8, characterized in that, The concentration of kanamycin monoclonal antibody in the gold-labeled antibody of kanamycin is 10 μg / mL; the concentration of gentamicin monoclonal antibody in the gold-labeled antibody of gentamicin is 12 μg / mL; the concentration of neomycin monoclonal antibody in the gold-labeled antibody of neomycin is 10 μg / mL; and the concentration of spectinomycin monoclonal antibody in the gold-labeled antibody of spectinomycin is 12 μg / mL. The pH of the solution is adjusted by the amount of K2CO3 added. Specifically, the amounts added to each 1 mL of colloidal gold solution are: 10 μg of kanamycin monoclonal antibody and 12 μL of K2CO3; 12 μg of gentamicin monoclonal antibody and 8 μL of K2CO3; 10 μg of neomycin monoclonal antibody and 10 μL of K2CO3; and 12 μg of spectinomycin monoclonal antibody and 8 μL of K2CO3.

10. The use of the multiplex immunochromatographic test strip according to any one of claims 1-9 in the simultaneous detection of kanamycin, gentamicin, neomycin, and spectinomycin, characterized in that, The detection limits were all 5 ng / mL.

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